Long-lived gadolinium based tumor targeted imaging and therapy agents

ABSTRACT

Alkylphosphocholine analogs incorporating a chelating moiety that is chelated to gadolinium are disclosed herein. The alkylphophocholine analogs are compounds having the formula: 
                         
or a salt thereof. R 1  includes a chelating agent that is chelated to a gadolinium atom; a is 0 or 1; n is an integer from 12 to 30; m is 0 or 1; Y is —H, —OH, —COOH, —COOX, —OCOX, or —OX, wherein X is an alkyl or an arylalkyl; R 2  is —N + H 3 , —N + H 2 Z, —N + HZ 2 , or —N + Z 3 , wherein each Z is independently an alkyl or an aroalkyl; and b is 1 or 2. The compounds can be used to detect solid tumors or to treat solid tumors. In detection/imaging applications, the gadolinium emits signals that are detectable using magnetic resonance imaging. In therapeutic treatment, the gadolinium emits tumor-targeting charged particles when exposed to epithermal neutrons.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. provisional Application No.62/252,218 filed on Nov. 6, 2015, which is incorporated by referenceherein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable.

FIELD OF THE DISCLOSURE

This disclosure relates generally to disease treatment and medicaldiagnosis/imaging. In particular, the disclosure is directed to (a)gadolinium-containing alkylphosphocholine analogs, (b) methods ofdetecting/imaging tumor cells using such compounds, and (c) relatedradiotherapy methods.

BACKGROUND

We have previously shown that certain alkylphosphocholine analogs arepreferentially taken up and retained by malignant solid tumor (i.e.,solid tumor cancer) cells. In U.S. Patent Publication No. 2014/0030187,which is incorporated by reference herein in its entirety, Weichert etal. disclose using analogs of the base compound18-(p-iodophenyl)octadecyl phosphocholine (NM404; see FIG. 1) fordetecting and locating, as well as for treating, a wide variety of solidtumor cancers. For example, if the iodo moiety is an imaging-optimizedradionuclide, such as iodine-124 ([¹²⁴I]-NM404), the analog can be usedin positron emission tomography-computed tomography (PET/CT) orsingle-photon emission computed tomography (SPECT) imaging of adultsolid tumors. Alternatively, if the iodo moiety is a radionuclideoptimized for delivering therapeutic doses of radiation to the solidtumors cells in which the analog is taken up, such as iodine-125 oriodine-131 ([¹²⁵I]-NM404 or [¹³¹I]-NM404), the analog can be used totreat solid tumors.

However, there are currently no long-lived computerized tomography (CT)or magnetic resonance (MR) imaging agents that have been shown tosuccessfully target tumor cells in vivo. Non-specific short-lived agentsin both modalities are used for cancer imaging by contrasting normalorgan tissues while in the process of renal or hepatobiliary excretion.There are currently a variety of radiopharmaceuticals available fortumor imaging, but these are limited by non-specificity for malignancy,the inability to distinguish cancer from inflammation, short biologicalhalf-life, and generally poor spatial resolution associated with PET andSPECT scanning modalities.

Accordingly, there is a need in the art for a tumor-specific agent foruse in MR scanning, CT scanning, or in both imaging methods. Such atumor-specific agent for MR or CT scanning would represent at least aten-fold improvement in the spatial resolution currently attainable withpositron emission agents and PET scanning.

BRIEF SUMMARY

The current disclosure provides new gadolinium (Gd)-labeled phospholipidcompounds that can be used long-lived tumor-specific MR imaging agentsand as neutron capture therapy agents.

The phospholipid metal chelate compounds disclosed herein utilize analkyl-phospholipid carrier combined with one of a variety of metalchelators that is chelated to a gadolinium atom. The disclosed metalchelates are preferentially taken up by malignant solid tumor cells, ascompared to non-tumor cells. Preferential uptake of such compoundsrenders them suitable for use as Gd-containing MR contrast/imagingagents that can be used in in malignant solid tumor detection/imagingapplications. Furthermore, the compounds can be used in therapeutictreatment, either by using the MR imaging results obtained using thecompounds as contrast agents for targeting external beam readiation toone or more solid tumors, or by using the compounds to facilitateneutron capture therapy against the tumors.

In a first aspect, the disclosure encompasses a compound having theformula:

or a salt thereof. R₁ includes or is a chelating agent that is chelatedto one or more gadolinium atoms; a is 0 or 1; n is an integer from 12 to30; m is 0 or 1; Y is —H, —OH, —COOH, —COOX, —OCOX, or —OX, wherein X isan alkyl or an arylalkyl; R₂ is —N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, or —N⁺Z₃, whereineach Z is independently an alkyl or an aroalkyl; and b is 1 or 2.

In some embodiments, the one or more gadolinium atoms are in the form ofa Gd(III) cation.

In some embodiments, the metal atom is an alpha, beta or Auger emittingmetal isotope with a half life of greater than 6 hours and less than 30days. Such isotopes are particularly suited for use in targetedradiotherapy applications. Non-limiting examples of such isotopesinclude Lu-177, Y-90, Ho-166, Re-186, Re-188, Cu-67, Au-199, Rh-105,Ra-223, Ac-225, As-211, Pb-212, and Th-227.

In some embodiments, the chelating agent is1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A) or one of itsderivatives; 1,4,7-triazacyclononane-1,4-diacetic acid (NODA) or one ofits derivatives; 1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA) orone of its derivatives;1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) or oneof its derivatives; 1,4,7-triazacyclononane, 1-glutaricacid-4,7-diacetic acid (NODAGA) or one of its derivatives;1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA) or one of its derivatives;1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid (TETA) orone of its derivatives;1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid (CB-TE2A)or one of its derivatives; diethylene triamine pentaacetic acid (DTPA),its diester, or one of its derivatives; 2-cyclohexyl diethylene triaminepentaacetic acid (CHX-A″-DTPA) or one of its derivatives; deforoxamine(DFO) or one of its derivatives;1,2-[[6-carboxypyridin-2-yl]methylamino]ethane (H₂dedpa) or one of itsderivatives; or DADA or one of its derivatives, wherein DADA has thestructure:

In some embodiments, a is 1 (aliphatic aryl-alkyl chain). In otherembodiments, a is 0 (aliphatic alkyl chain).

In some embodiments, m is 1 (acylphospholipid series).

In some embodiments, n is an integer between 12 and 20.

In some embodiments, Y is —OCOX, —COOX or —OX. In some such embodiments,X is —CH₂CH₃ or —CH₃.

In some embodiments, m is 0 (alkylphospholipid series).

In some embodiments, b is 1.

In some embodiments, n is 18.

In some embodiments, R₂ is —N⁺Z₃. In some such embodiments, each Z isindependently —CH₂CH₃ or —CH₃. In some such embodiments, each Z is —CH₃.

Non-limiting examples of the chelating agent that can be chelated to themetal atom include:

Non-limiting examples of the disclosed imaging and/or therapeutic agentsinclude:

In each case, the exemplary compound is chelated to one or moregadolinium atoms.

In some embodiments, the compound is:

In a second aspect, the disclosure encompasses a composition thatincludes one or of the compounds described above, and a pharmaceuticallyacceptable carrier.

In a third aspect, the disclosure encompasses one or more of thecompounds described above for use in magnetic resonance imaging ofcancer or cancerous cells.

In a fourth aspect, the disclosure encompasses one or more of thecompounds described above for use in treating cancer by neutron capturetherapy.

In a fifth aspect, the disclosure encompasses one or more of thecompounds described above for use in manufacturing a medicament fortreating or imaging cancer.

In a sixth aspect, the disclosure encompasses a method for detecting orimaging one or more cancer tumor cells in a biological sample. Themethod includes the steps of (a) contacting the biological sample withone or more of the compounds described above, whereby the compound isdifferentially taken up by malignant solid tumor cells within thebiological sample; and (b) identifying individual cells or regionswithin the biological sample that are emitting signals characteristic ofgadolinium.

In some embodiments, the step of identifying individual cells or regionswithin the biological sample that are emitting signals characteristic ofgadolinium is performed by magnetic resonance imaging (MRI).

In some embodiments, the biological sample is part or all of a subject.

In some embodiments, the biological sample is obtained from a subject.

In some embodiments, the subject is a human.

In some embodiments, the cancer cells are adult solid tumor cells orpediatric solid tumor cells. Non-limiting examples of such cells includemelanoma cells, neuroblastoma cells, lung cancer cells, adrenal cancercells, colon cancer cells, colorectal cancer cells, ovarian cancercells, prostate cancer cells, liver cancer cells, subcutaneous cancercells, squamous cell cancer cells, intestinal cancer cells,retinoblastoma cells, cervical cancer cells, glioma cells, breast cancercells, pancreatic cancer cells, Ewings sarcoma cells, rhabdomyosarcomacells, osteosarcoma cells, retinoblastoma cells, Wilms' tumor cells, andpediatric brain tumor cells.

In a seventh aspect, the disclosure encompasses a method of diagnosingcancer in a subject. The method includes one or more of theimaging/detection steps outlined above. In the method, the biogicalsample is obtained from, part of, or all of a subject. If cancer cellsare detected or imaged in the method steps, the subject is diagnosedwith cancer.

In some embodiments, the cancer that is diagnosed is an adult solidtumor or a pediatric solid tumor. Non-limiting examples of such cancerinclude melanoma, neuroblastoma, lung cancer, adrenal cancer, coloncancer, colorectal cancer, ovarian cancer, prostate cancer, livercancer, subcutaneous cancer, squamous cell cancer, intestinal cancer,retinoblastoma, cervical cancer, glioma, breast cancer, pancreaticcancer, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma,Wilms' tumor, and pediatric brain tumors.

In an eighth aspect, the disclosure encompasses a method of monitoringthe efficacy of a cancer therapy in a human subject. The method includesperforming one or more of the imaging/detection steps outlined above attwo or more different times on the biological sample, wherein thebiogical sample is obtained from, part of, or all of a subject. Thechange in strength of the signals characteristic of the metal isotopebetween the two or more different times is correlated with the efficacyof the cancer therapy.

In some embodiments, the cancer therapy being monitored is chemotherapyor radiotherapy.

In a ninth aspect, the disclosure encompasses a method of treatingcancer in a subject. The method includes performing one or more of theimaging/detection steps outlined above, wherein the biogical sample ispart of or all of a subject. The method also includes the step ofdirecting an external radiotherapy beam to the identified individualcells or regions within the subject.

In a tenth aspect, the disclosure encompasses a method for treating acancer in a subject by neutron capture therapy. The method includes thesteps of administering to a subject having cancer an effective amount ofone or more of the compounds described above, and radiating the subjectwith epithermal neutrons. The compounds absorb the neutrons andsubsequently emit high-energy charged particles to the localenvironment, which can effectively treat the cancer.

In some embodiments, the compound is administered by parenteral,intranasal, sublingual, rectal, or transdermal delivery. In some suchembodiments, the compound is administered intravenously. In someembodiments, the compound is administered intratumoraly.

In some embodiments, the subject is a human.

In some embodiments, the cancer that is treated is an adult solid tumoror a pediatric solid tumor. Non-limiting examples of cancers that couldbe treated include melanoma, neuroblastoma, lung cancer, adrenal cancer,colon cancer, colorectal cancer, ovarian cancer, prostate cancer, livercancer, subcutaneous cancer, squamous cell cancer, intestinal cancer,retinoblastoma, cervical cancer, glioma, breast cancer, pancreaticcancer, Ewings sarcoma, rhabdomyosarcoma, osteosarcoma, retinoblastoma,Wilms' tumor, and pediatric brain tumors.

Other objects, features and advantages of the present invention willbecome apparent after review of the specification, claims and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the chemical structure of the base compound18-(p-iodophenyl)octadecyl phosphocholine (NM404).

FIG. 2 shows a time course MRI image of a tumor-bearing mouse followinginjection of Gd-DO3A-404 showing enhancement of the tumor (T) by 24hours.

FIG. 3 (left panel) shows R₁ relaxation rate as a function ofGd-DO3A-404 concentration, both as T₁-weighted images (top left) andplotted as a graph (bottom left). The linear relationship shown by thegraph defines longitudinal relaxivity (r₁). FIG. 3 (right panel) showsR₂ relaxation rate as a function of Gd-DO3A-404 concentration, both asT₁-weighted images (top right) and plotted as a graph (bottom right).The linear relationship shown by the graph defines transverse relaxivity(r₂).

FIG. 4 shows time course MRI images of tumor-bearing mice. The top panelincludes images of a mouse bearing a flank A549 (human NSCLC) tumorbefore contrast agent injection (left, arrow showing tumor location),one hour after injection of Gd-DO3A-404 (second from left), 24 hoursfollowing injection of Gd-DO3A-404 (third from left), and 48 hoursfollowing injection of Gd-DO3A-404 (rightmost image). The bottom panelincludes images of a mouse bearing a flank U87 (human glioma) tumorbefore contrast agent injection (leftmost image, arrow showing tumorlocation), one hour after injection of Gd-DO3A-404 (second from left),24 hours following injection of Gd-DO3A-404 (third from left), and 48hours following injection of Gd-DO3A-404 (rightmost image).

FIG. 5 shows further time course MRI images of tumor-bearing mice,continuing from FIG. 4. The top panel includes images of the mousebearing a flank A549 (human NSCLC) tumor three days after injection ofGd-DO3A-404 (leftmost image), four days following injection ofGd-DO3A-404 (second from left), and seven days following injection ofGd-DO3A-404 (rightmost image). The bottom panel includes images of themouse bearing a flank U87 (human glioma) tumor three days afterinjection of Gd-DO3A-404 (leftmost image), four days following injectionof Gd-DO3A-404 (second from left), and seven days following injection ofGd-DO3A-404 (rightmost image).

FIG. 6 is a bar graph of quantified results from the images shown inFIGS. 4 and 5. Specifically, the tumor to muscle T1-weighted signalratios are shown for both the mouse bearing a flank A549 (human NSCLC)tumor (shaded bar) and the mouse bearing a flank U87 (human glioma)tumor (unshaded bar) before contrast agent injection (pre), one hourafter injection of Gd-DO3A-404, 24 hours after injection of Gd-DO3A-404,48 hours after injection of Gd-DO3A-404, three days after injection ofGd-DO3A-404, four days after injection of Gd-DO3A-404, and seven daysafter injection of Gd-DO3A-404. *p<0.05 compared to pre-contrast, A549.^(#)p<0.05 compared to pre-contrast, U87.

FIG. 7 is a bar graph of quantified results from the images shown inFIGS. 4 and 5. Specifically, the tumor to muscle R₁ ratios are shown forboth the mouse bearing a flank A549 (human NSCLC) tumor (shaded bar) andthe mouse bearing a flank U87 (human glioma) tumor (unshaded bar) beforecontrast agent injection (pre-contrast), and 48 hours after injection ofGd-DO3A-404. *p<0.05 compared to pre-contrast, A549. ^(#)p<0.05 comparedto pre-contrast, U87.

FIGS. 8, 9, 10, 11 and 12 are T1-weighted spoiled gradient (SPGR)magnetic resonance (MR) images of three different mouse abdomencross-sections, showing in vivo biodistribution of the Gd-DO3A-404contrast agent.

FIG. 8 includes T1-weighted SPGR MR images obtained before the contrastagent is injected. The locations of the myocardium (M, top image), liver(L, center image), and kidney (K, bottom image) are indicated by arrows,and are consistent with the corresponding images shown in FIGS. 9-12.

FIG. 9 includes T1-weighted SPGR MR images obtained one hour afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 10 includes T1-weighted SPGR MR images obtained 24 hours afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 11 includes T1-weighted SPGR MR images obtained four days afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 12 includes T1-weighted SPGR MR images obtained seven days afterGd-DO3A-404 contrast agent is injected. The images include themyocardium (top image), liver (center image), and kidney (bottom image).

FIG. 13 shows a time course MRI image of tumor-bearing (U87) mice before(pre) and for various times following injection of DOTA-chelated Gd³⁺(DOTAREM®, top panel) and Gd-DO3A-404 (bottom panel). Tumor location inthe mouse flank is indicated by the arrow in the two “pre” images.

FIG. 14 is a bar graph is a bar graph of quantified results from theimages shown in FIG. 13. Specifically, the tumor to muscle signal ratiosare shown for both the U87 mouse before (pre) and at various times afterinjection with DOTAREM® (shaded bars) or Gd-DO3A-404 (unshaded bars).*p<0.05 compared to pre-contrast, DOTAREM®. ^(#)p<0.05 compared topre-contrast, Gd-DO3A-404.

FIG. 15 shows MRI brain images of orthotopic glioblastoma model mice.2.5 mg (top panel) or 3.7 mg (bottom panel) of Gd-DOA3A-404 wasadministered to the mice by intravenous injection, and these images wereobtained 48 hours after contrast agent injection.

FIG. 16 is a bar graph showing tissue biodistribution of Gd-DO3A-404 inxenograft A549-flank bearing mice 72 hours post-administration. n=3mice.

FIG. 17 shows time course MRI images obtained from a transgenic mousetriple-negative breast cancer model (n=4; Animals/rows 1-4). Alpha-betacrystalline overexpressing mice were imaged pre-administration (leftmostcolumn) and 24 hours (center column) and 48 hours (rightmost column)post-administration.

FIG. 18 shows T₁-weighted images obtained from orthotopic xenograftmouse models. NOD-SCID mice with orthotopic U87 xenografts were imagedpre-administration, 24 hours, and 48 hours post administration ofGd-DO3A-404 (left panel). Orthotopic GSC 115 was imaged at 24 hours postadministration (right panel). GSC is a human glioma stem cell modelwhich was isolated from a human glioma patient.

FIG. 19 shows T₁-weighted scans of a U87 flank xenograft bearing ratusing a clinical 3.0 T PET/MR. Rats were imaged pre- and 24 hourspost-administration of Gd-DO3A-404.

FIG. 20 shows simultaneous PET/MR images of a U87-flank bearing rat 24hours post-administration of Gd-DO3A-404 and Cu-DO3A-404. Gd-DO3A-404and ⁶⁴Cu-DO3A-404 and were simultaneously administered to a U87-flankbearing rat. The rat was imaged using simultaneous PET/MR. Arrow pointsto tumor.

DETAILED DESCRIPTION

I. In General

It is understood that this disclosure is not limited to the particularmethodology, protocols, materials, and reagents described, as these mayvary. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present invention, which will be limited only by any later-filednonprovisional applications.

As used herein and in the appended claims, the singular forms “a”, “an”,and “the” include plural reference unless the context clearly dictatesotherwise. As well, the terms “a” (or “an”), “one or more” and “at leastone” can be used interchangeably herein. The terms “comprising” andvariations thereof do not have a limiting meaning where these termsappear in the description and claims. Accordingly, the terms“comprising”, “including”, and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. All publications and patentsspecifically mentioned herein are incorporated by reference for allpurposes including describing and disclosing the chemicals, instruments,statistical analysis and methodologies which are reported in thepublications which might be used in connection with the invention. Allreferences cited in this specification are to be taken as indicative ofthe level of skill in the art.

The terminology as set forth herein is for description of theembodiments only and should not be construed as limiting of theinvention as a whole. Unless otherwise specified, “a,” “an,” “the,” and“at least one” are used interchangeably and mean one or more than one.

The disclosure is inclusive of the compounds described herein (includingintermediates) in any of their pharmaceutically acceptable forms,including isomers (e.g., diastereomers and enantiomers), tautomers,salts, solvates, polymorphs, prodrugs, and the like. In particular, if acompound is optically active, the invention specifically includes eachof the compound's enantiomers as well as racemic mixtures of theenantiomers. It should be understood that the term “compound” includesany or all of such forms, whether explicitly stated or not (although attimes, “salts” are explicitly stated).

“Pharmaceutically acceptable” as used herein means that the compound orcomposition or carrier is suitable for administration to a subject toachieve the treatments described herein, without unduly deleterious sideeffects in light of the necessity of the treatment.

The term “effective amount,” as used herein, refers to the amount of thecompounds or dosages that will elicit the biological or medical responseof a subject, tissue or cell that is being sought by the researcher,veterinarian, medical doctor or other clinician.

As used herein, “pharmaceutically-acceptable carrier” includes any andall dry powder, solvents, dispersion media, coatings, antibacterial andantifungal agents, isotonic agents, absorption delaying agents, and thelike. Pharmaceutically-acceptable carriers are materials, useful for thepurpose of administering the compounds in the method of the presentinvention, which are preferably non-toxic, and may be solid, liquid, orgaseous materials, which are otherwise inert and pharmaceuticallyacceptable, and are compatible with the compounds of the presentinvention. Examples of such carriers include, without limitation,various lactose, mannitol, oils such as com oil, buffers such as PBS,saline, polyethylene glycol, glycerin, polypropylene glycol,dimethylsulfoxide, an amide such as dimethylacetamide, a protein such asalbumin, and a detergent such as Tween 80, mono- andoligopolysaccharides such as glucose, lactose, cyclodextrins and starch.

The term “administering” or “administration,” as used herein, refers toproviding the compound or pharmaceutical composition of the invention toa subject suffering from or at risk of the diseases or conditions to betreated or prevented.

A route of administration in pharmacology is the path by which a drug istaken into the body. Routes of administration may be generallyclassified by the location at which the substance is applied. Commonexamples may include oral and intravenous administration. Routes canalso be classified based on where the target of action is. Action may betopical (local), enteral (system-wide effect, but delivered through thegastrointestinal tract), or parenteral (systemic action, but deliveredby routes other than the GI tract), via lung by inhalation.

A topical administration emphasizes local effect, and substance isapplied directly where its action is desired. Sometimes, however, theterm topical may be defined as applied to a localized area of the bodyor to the surface of a body part, without necessarily involving targeteffect of the substance, making the classification rather a variant ofthe classification based on application location. In an enteraladministration, the desired effect is systemic (non-local), substance isgiven via the digestive tract. In a parenteral administration, thedesired effect is systemic, and substance is given by routes other thanthe digestive tract.

Non-limiting examples for topical administrations may includeepicutaneous (application onto the skin), e.g., allergy testing ortypical local anesthesia, inhalational, e.g. asthma medications, enema,e.g., contrast media for imaging of the bowel, eye drops (onto theconjunctiva), e.g., antibiotics for conjunctivitis, ear drops, such asantibiotics and corticosteroids for otitis externa, and those throughmucous membranes in the body.

Enteral administration may be administration that involves any part ofthe gastrointestinal tract and has systemic effects. The examples mayinclude those by mouth (orally), many drugs as tablets, capsules, ordrops, those by gastric feeding tube, duodenal feeding tube, orgastrostomy, many drugs and enteral nutrition, and those rectally,various drugs in suppository.

Examples of parenteral administrations may include intravenous (into avein), e.g. many drugs, total parenteral nutrition intra-arterial (intoan artery), e.g., vasodilator drugs in the treatment of vasospasm andthrombolytic drugs for treatment of embolism, intraosseous infusion(into the bone marrow), intra-muscular, intracerebral (into the brainparenchyma), intracerebroventricular (into cerebral ventricular system),intrathecal (an injection into the spinal canal), and subcutaneous(under the skin). Among them, intraosseous infusion is, in effect, anindirect intravenous access because the bone marrow drains directly intothe venous system. Intraosseous infusion may be occasionally used fordrugs and fluids in emergency medicine and pediatrics when intravenousaccess is difficult.

As used herein, the term “intraperitoneal injection” or “IP injection”refers to the injection of a substance into the peritoneum (bodycavity). IP injection is more often applied to animals than to humans.In general, IP injection may be preferred when large amounts of bloodreplacement fluids are needed, or when low blood pressure or otherproblems prevent the use of a suitable blood vessel for intravenousinjection.

II. The Invention

In certain aspects, the disclosure is directed to the use ofgadolinium-labeled alkylphosphocholine analogs for medical detection ordetection/imaging of cancerous tumors or tumor cells in a subject or ina biological sample. In other aspects, the diclosure is directed to theuse of gadolinium-labeled alkylphosphocholine analogs to treat cancer ina subject. In yet other aspects, the diclosure is directed to thegadolinium-labeled alkylphosphocholine analogs and methods ofsynthesizing such compounds.

A. Neutron Capture Therapy

Neutron capture therapy (NCT) is a non-invasive therapeutic method fortreating locally invasive malignant tumors, such as primary brain tumorsand recurrent head and neck cancer. In NCT, the patient is firstinjected with a tumor localizing drug containing a non-radioactiveisotope that has a high propensity or cross section (σ) to capture slowneutrons (the “capture agent”). The cross section of the capture agentis many times greater than that of the other elements present intissues, such as hydrogen, oxygen, and nitrogen. In the second step, thepatient is radiated with epithermal neutrons, which after losing energyas they penetrate tissue, are absorbed by the capture agent. The captureagent subsequently emits high-energy charged particles that caneffectively kill cancerous tissue.

All of the clinical experience to date with NCT uses the non-radioactiveisotope boron-10 as the capture agent. However, the Gd-labeled PLEanalogs disclosed herein may be ideal neutron capture therapy agents,since these compounds exhibit malignant tumor selectivity, and ¹⁵⁷Gd hasthe highest thermal neutron cross section of any stable nucleotide,namely 25900 barn, which 8 times that of Boron.

B. MRI Detection/Imaging

The disclosed compounds are the first long-lived tumor-specific MRcontrast agent for general broad spectrum tumor imaging andcharacterization. In addition, the compounds the long-livedtumor-specific MR contrast agents disclosed herein are suitable for usein therapy response monitoring to both chemotherapy and radiotherapy,and are suitable tumor contrast agent for MRI guided external beamradiotherapy.

A course of cancer radiotherapy may extend over a time frame of 5 to 7weeks. Therapy is delivered daily, making the administration ofshort-lived MR contrast agents impractical, due to their renal toxicity.Therefore, a long-lived tumor specific contrast agent that has abiological half-life of weeks rather than minutes to hours (e.g., thedisclosed compounds) may be ideal for external beam radiotherapy.

The contrast agent would be administered a few days before the start oftreatment simulation to allow for adequate accumulation of the agent inthe tumor. Boost doses may have to be administered either weekly orbiweekly during the course of therapy. An MRI simulation would becarried out and a treatment plan would be developed. The patient wouldthen be treated using an MRI guided radiotherapy system, which are nowbecoming commercially available, and the long-lived tumor-targetedcontrast agent would then be used to optimize the dose delivery and totrack the tumor tracking during therapy delivery, since these MRI guidedradiotherapy systems allow for fast intratreatment imaging.

Using such methods, a moving tumor can be tracked using such an agentover the course of therapy. However, as pointed out above, the utilityof the disclosed long-lived tumor specific MRI contrast imaging agentgoes far beyond radiotherapy. Such an imaging agent can also be employedfor response monitoring of chemotherapy as well as radiotherapy. Forthis to be feasible one indeed needs long-lived tumor specific imagingagents that will be retained in tumor cells. As tumor cells die throughapoptosis or undergo mitotic catastrophe, the imaging agent is releasedfrom the tumor cells, leading to changes in the resulting MRI signalthat allow for assessment of therapy response.

C. Gadolinium-Labeled PLE Analogs

The disclosed structures utilize an alkylphosphocholine carrierbackbone. Once synthesized, the agents must harbor formulationproperties that render them suitable for injection while retaining tumorselectivity. A non-limiting exemplary series of Gd-PLE analogs follows(additional non-limiting examples were described previously). Thestructures shown include a chelating moiety to which the gadolinium ionis chelated to produce the final imaging or therapeutic agent.

D. Methods of Synthesizing Exemplary Gd-PLE Analogs

Proposed synthesis of compound 1 is shown below. The first step of thesynthesis is similar to described in Org Synth, 2008, 85, 10-14. Thesynthesis is started from cyclen which is converted into DO3A tris-Bnester. This intermediate is then conjugated with NM404 in the presenceof the base and Pd catalyst. Finally, benzyl protecting groups areremoved by the catalytic hydrogenation.

Synthesis of compound 2 is shown below. It begins with DO3A tris-Bnester which is alkylated with 3-(bromo-prop-1-ynyl)-trimethylsilane.After alkylation, the trimethylsilyl group is removed and theintermediate acetylene is coupled with NM404 by the Sonogashirareaction. The benzyl groups are removed and the triple bond ishydrogenated simultaneously in the last step of the synthesis.

Compounds 5 and 6 can be synthesized from same precursors, DTPAdianhydride and 18-p-(3-hydroxyethyl-phenyl)-octadecyl phosphocholine asshown in the schemes below.

NOTA-NM404 conjugates can be synthesized in an analogous manner. Onenon-limiting example is NOTA-NM404 conjugate 7:

E. Dosage Forms and Administration Methods

Any route of administration may be suitable for administering thedisclosed Gd-PLE analogs to a subject. In one embodiment, the disclosedanalogs may be administered to the subject via intravenous injection. Inanother embodiment, the disclosed analogs may be administered to thesubject via any other suitable systemic deliveries, such as oral,parenteral, intranasal, sublingual, rectal, or transdermaladministrations.

In another embodiment, the disclosed analogs may be administered to thesubject via nasal systems or mouth through, e.g., inhalation.

In another embodiment, the disclosed analogs may be administered to thesubject via intraperitoneal injection or IP injection.

In certain embodiments, the disclosed analogs may be provided aspharmaceutically acceptable salts. Other salts may, however, be usefulin the preparation of the analogs or of their pharmaceuticallyacceptable salts. Suitable pharmaceutically acceptable salts include,without limitation, acid addition salts which may, for example, beformed by mixing a solution of the analog with a solution of apharmaceutically acceptable acid such as hydrochloric acid, sulphuricacid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid,acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid,carbonic acid or phosphoric acid.

Where the disclosed analogs have at least one asymmetric center, theymay accordingly exist as enantiomers. Where the disclosedalkylphosphocholine analogs possess two or more asymmetric centers, theymay additionally exist as diastereoisomers. It is to be understood thatall such isomers and mixtures thereof in any proportion are encompassedwithin the scope of the present disclosure.

The disclosure also includes methods of using pharmaceuticalcompositions comprising one or more of the disclosed analogs inassociation with a pharmaceutically acceptable carrier. Preferably thesecompositions are in unit dosage forms such as tablets, pills, capsules,powders, granules, sterile parenteral solutions or suspensions, meteredaerosol or liquid sprays, drops, ampoules, auto-injector devices orsuppositories; for oral, parenteral, intranasal, sublingual or rectaladministration, or for administration by inhalation or insufflation.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutically acceptable carrier, e.g.conventional tableting ingredients such as corn starch, lactose,sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalciumphosphate or gums, and other pharmaceutical diluents, e.g. water, toform a solid preformulation composition containing a homogeneous mixturefor a compound of the present invention, or a pharmaceuticallyacceptable salt thereof. The liquid forms in which the analogs may beincorporated for administration orally or by injection include aqueoussolutions, suitably flavored syrups, aqueous or oil suspensions, andflavored emulsions with edible oils such as cottonseed oil, sesame oil,coconut oil or peanut oil, as well as elixirs and similar pharmaceuticalvehicles. Suitable dispersing or suspending agents for aqueoussuspensions include synthetic and natural gums such as tragacanth,acacia, alginate, dextran, sodium caboxymethylcellulose,methylcellulose, polyvinylpyrrolidone or gelatin.

The disclosed analogs are particularly useful when formulated in theform of a pharmaceutical injectable dosage, including in combinationwith an injectable carrier system. As used herein, injectable andinfusion dosage forms (i.e., parenteral dosage forms) include, but arenot limited to, liposomal injectables or a lipid bilayer vesicle havingphospholipids that encapsulate an active drug substance. Injectionincludes a sterile preparation intended for parenteral use.

Five distinct classes of injections exist as defined by the USP:emulsions, lipids, powders, solutions and suspensions. Emulsioninjection includes an emulsion comprising a sterile, pyrogen-freepreparation intended to be administered parenterally. Lipid complex andpowder for solution injection are sterile preparations intended forreconstitution to form a solution for parenteral use. Powder forsuspension injection is a sterile preparation intended forreconstitution to form a suspension for parenteral use. Powderlyophilized for liposomal suspension injection is a sterile freeze driedpreparation intended for reconstitution for parenteral use that isformulated in a manner allowing incorporation of liposomes, such as alipid bilayer vesicle having phospholipids used to encapsulate an activedrug substance within a lipid bilayer or in an aqueous space, wherebythe formulation may be formed upon reconstitution. Powder lyophilizedfor solution injection is a dosage form intended for the solutionprepared by lyophilization (“freeze drying”), whereby the processinvolves removing water from products in a frozen state at extremely lowpressures, and whereby subsequent addition of liquid creates a solutionthat conforms in all respects to the requirements for injections. Powderlyophilized for suspension injection is a liquid preparation intendedfor parenteral use that contains solids suspended in a suitable fluidmedium, and it conforms in all respects to the requirements for SterileSuspensions, whereby the medicinal agents intended for the suspensionare prepared by lyophilization. Solution injection involves a liquidpreparation containing one or more drug substances dissolved in asuitable solvent or mixture of mutually miscible solvents that issuitable for injection.

Solution concentrate injection involves a sterile preparation forparenteral use that, upon addition of suitable solvents, yields asolution conforming in all respects to the requirements for injections.Suspension injection involves a liquid preparation (suitable forinjection) containing solid particles dispersed throughout a liquidphase, whereby the particles are insoluble, and whereby an oil phase isdispersed throughout an aqueous phase or vice-versa. Suspensionliposomal injection is a liquid preparation (suitable for injection)having an oil phase dispersed throughout an aqueous phase in such amanner that liposomes (a lipid bilayer vesicle usually containingphospholipids used to encapsulate an active drug substance either withina lipid bilayer or in an aqueous space) are formed. Suspension sonicatedinjection is a liquid preparation (suitable for injection) containingsolid particles dispersed throughout a liquid phase, whereby theparticles are insoluble. In addition, the product may be sonicated as agas is bubbled through the suspension resulting in the formation ofmicrospheres by the solid particles.

The parenteral carrier system includes one or more pharmaceuticallysuitable excipients, such as solvents and co-solvents, solubilizingagents, wetting agents, suspending agents, thickening agents,emulsifying agents, chelating agents, buffers, pH adjusters,antioxidants, reducing agents, antimicrobial preservatives, bulkingagents, protectants, tonicity adjusters, and special additives.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.Indeed, various modifications of the invention in addition to thoseshown and described herein will become apparent to those skilled in theart from the foregoing description and the following examples and fallwithin the scope of the appended claims.

III. Examples

Summary:

In Example 1, we provide an exemplary synthesis that also finds use forthe synthesis of analogous compounds chelating radioactive metalisotopes.

In Example 2, we demonstrate that an analog having a chelating agent andchelated metal substituted for the iodine moiety of NM404 (Gd-DO3A-404)is taken up by (and can be imaged in) solid tumor tissue, thus providingproof of concept for using the disclosed metal chelates as TRT and/orimaging agents.

In Example 3, we demonstrate the stability of Gd-DO3A-404.

In Example 4, we characterize the magnetic resonance (MR) relaxationcharacteristics (r₁ and r₂) of Gd-DO3A-404.

In Example 5, we extended the results of Example 2 to demonstrate thetumor-targeting capabilities and uptake dynamics of Gd-DO3A-404 in twodifferent tumor models.

In Example 6, we report in-vivo biodistribution data for Gd-DO3A-404.

In Example 7, we demonstrate that the tumor-targeting properties ofGd-DO3A-404 reside in the NM404 targeting moiety. Specifically, wecompare the tumor uptake and retention data for Gd-DO3A-404 with thesame data obtained using DOTA-chelated Gd³⁺ (DOTAREM®).

In Example 8, we demonstrate Gd-DO3A-404 uptake in an orthotopicglioblastoma model.

In Example 9, we disclose biodistribution data for Gd-DOA-404 afterbeing administered to flank A549 xenograft mice.

In Example 10, we demonstrate Gd-DO3A-404 uptake in a triple-negativebreast cancer model.

In Example 11, we demonstrate Gd-DO3A-404 uptake in two orthotopicxenograft models.

In Example 12, we demonstrate simultaneous uptake and imaging (PET andMRI) of the gadolinium chelate Gd-DO3A-404, acting as an MRI contrastagent, and the copper radionuclide Cu-64 chelate ⁶⁴Cu-DO3A-404, whichacts as a PET contrast agent.

Example 1 Synthesis of Metal Chelated DO3A-404

In this Example, we show the synthetic scheme used to synthesize oneexemplary phospholipid chelate, Gd-DO3A-404.

Example 2 In Vivo Imaging Proof of Concept

In this example, we demonstrate the successful in vivo MRI imaging of atumor, using Gd-DO3A-404 as the MRI contrast agent.

For proof-of-concept in vivo imaging of tumor uptake of the Gd-DO3A-404agent, nude athymic mouse with a flank A549 tumor (non small cell lungcancer) xenograft was scanned. The Gd-DO3A-404 agent (2.7 mg) wasdelivered via tail vein injection. Mice were anesthetized and scanningperformed prior to contrast administration and at 1, 4, 24, 48, and 72hours following contrast delivery. Imaging was performed on a 4.7 TVarian preclinical MRI scanner with a volume quadrature coil.T1-weighted images were acquired at all imaging time points using a fastspin echo scan with the following pulse sequence parameters: repetitiontime (TR)=206 ms, echo spacing=9 ms, echo train length=2, effective echotime (TE)=9 ms, 10 averages, with a 40×40 mm² field of view, 192×192matrix, 10 slices of thickness 1 mm each.

As seen in FIG. 2, MRI imaging of the tumor was significantly enhancedby 24 hours post-injection.

These results demonstrate that the differential uptake and retention ofalkylphosphocholine analogs is maintained for the gadolinium chelatedanalogs disclosed herein. Thus, the disclosed gadolinium chelates canreadily be applied to clinical therapeutic and imaging applications.

Example 3 Stability of Gd-DO3A-404

In this example, we quantitated the free Gd concentration associatedwith Gd-DO3A-404. The results demonstrate that Gd-DO3A-404 is quitestable, and substantially retains the chelated Gd (III) ion. Thus, it issuitable for use as a tumor-targeting agent for use in imaging and/ortherapeutic applications.

We considered several methods that are conventionally employed toquantitate the the free Gd (III) ion concentration. First, we consideredusing chromophoric ligands. For example, using Xylenol Orange, freeGd(III) can be reliably detected down to concentrations of 1 μM. Thisdetection limit is too high to reliably detect free Gd in this context.The 5-Br-PADAP ligand provides a significantly lower detection limit,down to 0.1 μM Gd(III). However, we observed a detrimental interactionbetween the ligand and the Gd-DO3A-404 that rendered this methodunusable in this context.

Next, we considered using membrane-based separation approaches. However,such methods proved to be problematic in this context, due to retainmentof the free Gd by the membrane.

After considering other alternatives, we decided to determine free Gdconcentration by selective chelation and separation of the free Gd. Weseparated the Gd-DO3A-404 complex from free Gd (III) ion by CHELEX®solid phase extraction (as described by Raju, et al., J. Anal. At.Spectrom. 25 (2010), 1573-1580). Specifically, using a CHELEX® solidphase extraction column comprising an immobilized ligand(iminodiacetate) with very high affinity for free ion species ofmulti-valent transition and rare-earth metals, Gd was retained and theneluted using a known ratio of chelated Gd-DO3A-404 and acid-digestedGd-DO3A-404. The sample was the analyzed by magnetic sector ICP-MS todetermine the concentration of free Gd. The free Gd concentration wasfound to be 0.081%.

Example 4 Gd-DO3A-404 Shows Favorable Relaxivity for T₁-Weighted Imaging

In this example, we characterized the magnetic resonance relaxationcharacteristics of Gd-DO3A-404, which compare favorably to thecharacteristics of commonly used contrast agents.

For initial studies, relaxivity was measured at 4.7 T. Longitudinalrelaxivity, which is defined by the linear relationship between R₁relaxation rate and concentration, was measured using an inversionrecovery spin echo sequence.

Higher relaxivity results in brighter signal on a T₁-weighted image, andthus indicates greater T₁-weighted signal enhancement potential. Byplotting R₁ (=1/T₁) versus concentration at five differentconcentrations of the Gd-DOTA-404 agent and determining the equation ofthe resulting line (see FIG. 3, left panel), we calculated alongitudinal relaxivity in plasma of 5.74 s-1/mM. This comparesfavorably to clinical T₁ shortening contrast agents (such as DOTOAREM®),which have been shown to have relaxivities of 2-3 s-1/mM at this fieldstrength.

Similarly, by plotting R₂ versus concentration at five differentconcentrations of the Gd-DOTA-404 agent and determining the equation ofthe resulting line (see FIG. 3, right panel), we calculated a transverserelaxivity in plasma of 20.4 s-1/mM. Thus, r2 relaxivity is alsofavorably increased when using Gd-DOTA-404 as a contrast agent (see FIG.3, right panel).

Determination of r₁ of the agent in saline, excipient, and plasma showedthat it consistently resulted in shortened T₁ times. See Table 1.

TABLE 1 Longitudinal Relaxivity (r₁) and Transverse Relaxivity (r₂) ofGd-DO3A-404 Relaxivity of Gd-DO3A-404 (s⁻¹/mM) In saline In excipient Inplasma r₁ 5.68 5.84 5.74 r₂ 16.14 11.31 20.36

These data demonstrate that the disclosed gadolinium chelated analogswould be effective contrast agents for magnetic resonance imagingapplications.

Example 5 In Vivo Cancer Imaging in Multiple Tumor Models

In this extension of Example 2, we demonstrate selective uptake and invivo MRI imaging in two distinct flank tumor types, using Gd-DO3A-404 asthe MRI contrast agent.

To test uptake and retention in rodent models of human cancer, flankxenografts were established in mice for two distinct tumor types, A549(human non small cell lung cancer, NSCLC) and U87 (human glioma). N=3for each model. For pre-contrast imaging, T₁-W images of the tumor andabdomen (FIG. 4; 2 leftmost images) and T₁ maps of the tumor wereobtained.

At time zero (“contrast”), 2.5 mg of Gd-DO3A-404 (˜12 mmol/kg body mass)was delivered into the mice by intravenous injection. Animals werescanned pre-contrast and at various time points between one hour andseven days post-contrast (after one hour, 24 hours, 48 hours, threedays, four days and seven days). T₁ maps of the tumor were acquired foreach time point, along with T₁-weighted images of the tumor and theabdomen (see FIGS. 4 and 5).

In the NSCLC model, Gd-DO3A-404 uptake was not immediate and reached amaximum at 24-48 hours post-contrast (FIG. 4). The uptake was maintainedover several days (FIG. 5). In the U87 model, uptake was more rapid(already observable at one hour after delivery; see FIG. 4) and appearedto reach higher levels and was maintained for a longer time period (seeFIG. 5).

Those observations were confirmed by the quantified data, where tumor tomuscle T₁-weighted signal ratios were approximately doubled followingGd-DO3A-404 delivery (FIG. 6). The increase in tumor signal was morerapid and more prolonged in U87 tumors as compared to A549 tumors. Asseen in FIG. 7, the R₁ relaxation rate for both tumor types wassignificantly increased at 48 hours post-contrast.

These results demonstrate that the differential uptake and retention ofalkylphosphocholine analogs in multiple tumor types is maintained forthe gadolinium chelated analogs disclosed herein. Thus, the disclosedgadolinium chelates can readily be applied to clinical therapeutic andimaging applications.

Example 6 Use of MRI to Determine In Vivo Biodistribution of Gd-DO3A-404

In this example, we determined the in vivo biodistribution of theGd-DO3A-404 after the contrast agent was administered (see Example 5).During the course of performing the experiments described in Example 4,we also acquired T₁-weighted spoiled gradient (SPGR) images in theabdomen of the mice, to observe biodistribution. Abdominalcross-sections imaged included the myocardium (FIGS. 8-12, top image),the liver (FIGS. 8-12, center image), and a kidney (FIGS. 8-12, bottomimage). Images are shown pre-contrast (FIG. 8), and at one hour (FIG.9), 24 hours (FIG. 10), four days (FIG. 11) and seven days post-contrast(FIG. 12).

In the myocardium and blood pool, the Gd-DO3A-404 contrast agentcirculates for nearly up to a day, after which any signal observed isdue to retention rather than from further uptake. In the liver andkidney, the Gd-DO3A-404 contrast agent is substantially cleared overtime, with more rapid clearance occurring through the liver, and moreprolonged clearance occurring through the kidney. Notably, theGd-DO3A-404 contrast agent exhibits a P-kinetic profile, includinghepatobiliary excretion, that is similar to that of relatedalkylphosphocholine analogs.

Example 7 The Gd-DO3A-404 Targeting Moiety Facilitates Tumor-Selectiveand Sustained Uptake

In this example, we demonstrate that the selective uptake and retentionof Gd-DO3A-404 in tumor tissues is in fact facilitated by thetumor-targeting phospholipid moiety (the “404” moiety; see FIG. 1),rather than by the gadolinium metal or its chelating agent. Accordingly,this Example demonstrates that effective tumor-targeting contrast agentsare not limited to those having a specific chelating agent, as long asthey include the disclosed tumor-targeting phospholipid moieties.

To verify that uptake and retention was due to targeting of the “404”moiety, we directly compared the uptake of Gd-DO3A-404 with that ofDOTA-chelated Gd³⁺ (DOTAREM®) in an identical tumor model (mice withflank U87 tumors) and imaging scenario, using the same number of molesof each. As seen in FIG. 13, the uptake and clearance of DOTAREM®, ismuch more rapid than that of Gd-DO3A-404.

We the quantified the tumor to muscle ratio and compare it to baselinescans. As seen in FIG. 14, the DOTOREM® uptake was less striking, andsignificant only at a couple of early time points, as compared toGd-DO3A-404.

These results show that the phospholipid targeting moiety of Gd-DO3A-404(the 404 moiety), not the chelating agent and chelated metal (theGd-DO3A moiety) are responsible for the observed selective tumor uptakeand retention. Thus, a variety of different chelating moieties can beused without affecting the selective tumor uptake and retentionproperties of the disclosed chelates.

Example 8 Brain Tumor Uptake of Gd-DO3A-404 in Orthotopic Glioma Model

In this example, we demonstrate that at higher dosages, Gd-DO3A-404 canpass through the blood-brain barrier to successfully target brain tumortissue.

To investigate the use of Gd-DO3A-404 to detect tumors and metasteses insitu, in particular, in the brain, we created an orthotopic glioblastomamodel using cancer stem cells injected into the brain. To create themodel, brains of mice were injected with cells from orthotopicglioblastoma stem cell line 12.6. After sufficient tumor growth,monitored with T₂-weighted MRI, we imaged subjects pre-contrast andafter delivery (24-72 hours) of two different doses of Gd-DO3A-404 (2.5or 3.7 mg; ˜0.12-0.18 mmole/kg).

At the lower dose used for flank xenografts, no brain uptake wasobserved (see FIG. 15, upper panel). Because the lower delivered dosewas relatively low (on the same order of that delivered per kg bodyweight in clinical settings), we increased the dose for another group ofanimals. In this group, we observed uptake in one subject (FIG. 15,bottom panel). This result indicates that the blood-brain barrier (BBB)may be playing a role in brain tumor uptake, and dosage may be “tuned”to facilitate the contrast agent's passage through the BBB.

Example 9 In Vivo Biodistribution Data for Gd-DO3A-404 in Flank A549Xenograft Mice

In this extension of Example 6, we further examined the in vivobiodistribution of Gd-DO3A-404 after it is administered. Specifically,tissue biodistribution was measured in A549-flank bearing mice 72 hoursafter administration of Gd-DO3A-404. Nude athymic mice were sacrificed,perfused and tissues were collected and quantitated for Gd byhigh-resolution (magnetic-sector) inductively-coupled plasma massspectrometry (SF-ICPMS). n=3 mice.

As seen in FIG. 16, the Gd-DO3A-404 was selectively taken up by tumortissue, again demonstrating the suitability of the disclosedalkylphosphocholine analogs for targeted delivery to tumor tissue.

Example 10 Uptake of Gd-DO3A-404 in Triple-Negative Breast Cancer Model

In this example, we demonstrate the successful targeting of Gd-DO3A-404to breast cancer tissue.

Alpha-beta crystalline overexpressing mice (a triple negative breastcancer model) underwent MR imaging pre administration and 24 hours and48 hours post-administration of Gd-DO3A-400 (n=4). As seen in FIG. 17,over 48 hours, the contrast agent was taken up by and localized to thebreast cancer tissue.

This example illustrates that the disclosed alkylphosphocholine metalchelates can be used to target a wide range of solid tumor tissues.

Example 11 Uptake of Gd-DO3A-404 in Orthotopic Model

In this example, we demonstrate the successful targeting of Gd-DO3A-404in two different orthotopic xenograft models.

NOD-SCID mice with orthotopic U87 xenografts were imagedpre-administration, 24 hours, and 48 hours post administration ofGd-DO3A-404. As seen in FIG. 18 (left panel), the contrast agent wasdifferentially taken up by the tumor tissue (see arrows).

Orthotopic GSC 115 was imaged at 24 hours post administration ofGd-DO3A-404. GSC is a human glioma stem cell model which was isolatedfrom a human glioma patient. As seen in FIG. 18 (right panel), thecontrast agent was differentially taken up by the tumor tissue (seearrow).

This example illustrates that the disclosed alkylphosphocholine metalchelates can be used to target a wide range of solid tumor tissues.

Example 12 Simultaneous PET/MR Imaging Demonstrating Tumor Targeting byBoth Gd-DO3A-404 and ⁶⁴Cu-DO3A-404

In this example, we demonstrate the successful use of both Gd-DO3A-404and ⁶⁴Cu-DO3A-404 as tumor targeting contrast agents (Gd-DO3A-404 forMRI and ⁶⁴Cu-DO3A-404 for simultaneous PET imaging).

Simultaneous imaging was performed using a clinical Pet/MRI scanner.⁶⁴Cu-DO3A-404 has the same structure as Gd-DO3A-404, except that ⁶⁴Cu, apositron emitting radionuclide, is chelated to the chelating moietyinstead of Gd. ⁶⁴Cu-DO3A-404 was synthesized (and can be synthesizedusing the methods disclosed herein; see, e.g., Example 1). Both the⁶⁴Cu-DO3A-404 and Gd-DO3A-404 chelates were injected simultaneously intoa rat with a flank U87 (human glioma) xenograft.

T1-weighted scans of the U87 flank xenograft were obtained using theclinical 3.0 T PET/MR. Rats were imaged pre- and 24 hourspost-administration of the Gd-DO3A-404. The resulting MR imagesdemonstrate selective tumor uptake of the Gd-DO3A-404 contrast agent(FIG. 19; arrow showing tumor location).

Simultaneous PET/MR scans of the U87-flank bearing rat 24 hourspost-simultaneous administration of both Gd-DO3A-404 (the MRI contrastagent) and ⁶⁴Cu-DO3A-404 (the PET contrast agent) were obtained. As seenin FIG. 20, fused T1-weighted MR and PET images showed excellentcolocalization of contrast and activity in the flank and abdomen (arrowpoints to tumor). The tumor is enhanced enhances in both the T1 and T2MRI images (FIG. 20). Furthermore, the simultaneous PET scandemonstrates tumor uptake of the ⁶⁴Cu-DO3A-404 PET contrast agent (FIG.20), providing proof-of concept for using the disclosed chelates havinga radioactive metal substituted for Gd in tumor imaging (such as PETimaging) and radiotherapy applications.

In sum, these examples demonstrate that gadolinium metal chelates thatinclude an appropriate tumor-targeting phospholipid moiety, as disclosedherein, can be effective MRI contrast agents that demonstratesignal-enhancing uptake and retention in multiple cancer types. Suchcontrast agents will facilitate the detection, characterization, andstaging of cancer and metastases with high spatial resolution.Furthermore, due to the high neutron capture cross-section ofgadolinium, such agents may also have applications in targeted neutroncapture therapy of cancer.

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration from the specification andpractice of the invention disclosed herein. All references cited hereinfor any reason, including all journal citations and U.S./foreign patentsand patent applications, are specifically and entirely incorporatedherein by reference. It is understood that the invention is not confinedto the specific reagents, formulations, reaction conditions, etc.,herein illustrated and described, but embraces such modified formsthereof as come within the scope of the following claims.

The invention claimed is:
 1. A compound having the formula:

or a salt thereof, wherein: R₁ comprises a chelating agent that ischelated to one or more gadolinium (Gd) atoms; a is 0 or 1; n is aninteger from 12 to 30; m is 0 or 1; Y is selected from the groupconsisting of —H, —OH, —COOH, —COOX, —OCOX, and —OX, wherein X is analkyl or an arylalkyl; R₂ is selected from the group consisting of—N⁺H₃, —N⁺H₂Z, —N⁺HZ₂, and —N⁺Z₃, wherein each Z is independently analkyl or an aroalkyl; and b is 1 or 2 wherein the chelating agent isselected from the group consisting of1,4,7,10-tetraazacyclododecane-1,4,7-triacetic acid (DO3A);1,4,7-triazacyclononane-1,4-diacetic acid (NODA);1,4,7-triazacyclononane-1,4,7-triacetic acid (NOTA);1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA);1,4,7-triazacyclononane, 1-glutaric acid-4,7,10-diacetic acid (NODAGA);1,4,7,10-tetraazacyclodecane, 1-glutaric acid-4,7,10-triacetic acid(DOTAGA); 1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraacetic acid(TETA); 1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid(CB-TE2A); diethylene triamine pentaacetic acid (DTPA), its diester;2-cyclohexyl diethylene triamine pentaacetic acid (CHX-A″-DTPA);deforoxamine (DFO); 1,2-[[6-carboxypyridin-2-yl]methylamino]ethane(H₂dedpa); and DADA, wherein DADA has the structure:


2. The compound of claim 1, wherein the one or more gadolinium atoms arein the form of a Gd(III) ion.
 3. The compound of claim 1, wherein thechelating agent chelated to the gadolinium atom is selected from thegroup consisting of:


4. The compound of claim 1, wherein the compound is selected from thegroup consisting of:

wherein the selected compound is chelated to a gadolinium atom.
 5. Thecompound of claim 4, wherein the compound is:


6. A composition comprising a compound according to claim 1 and apharmaceutically acceptable carrier.
 7. A method for detecting orimaging one or more cancer tumor cells in a biological sample,comprising: (a) contacting the biological sample with a compound ofclaim 1; and (b) identifying individual cells or regions within thebiological sample that are emitting signals characteristic ofgadolinium, whereby one or more cancer tumor cells are detected orimaged.
 8. The method of claim 7, wherein the step of identifyingindividual cells or regions within the biological sample that areemitting signals characteristic of gadolinium is performed by magneticresonance imaging (MRI).
 9. The method of claim 7, wherein thebiological sample is part or all of a subject.
 10. The method of claim7, wherein the biological sample is obtained from a subject.
 11. Themethod of claim 9, wherein the subject is a human.
 12. The method ofclaim 7, wherein the cancer cells are adult solid tumor cells orpediatric solid tumor cells.
 13. The method of claim 12, wherein thecancer cells are selected from the group consisting of melanoma cells,neuroblastoma cells, lung cancer cells, adrenal cancer cells, coloncancer cells, colorectal cancer cells, ovarian cancer cells, prostatecancer cells, liver cancer cells, subcutaneous cancer cells, squamouscell cancer cells, intestinal cancer cells, retinoblastoma cells,cervical cancer cells, glioma cells, breast cancer cells, pancreaticcancer cells, Ewings sarcoma cells, rhabdomyosarcoma cells, osteosarcomacells, retinoblastoma cells, Wilms' tumor cells, and pediatric braintumor cells.
 14. A method of diagnosing cancer in a subject, comprisingperforming the method of claim 7, wherein the biological sample isobtained from, part of, or all of a subject, and whereby if cancer cellsare detected or imaged, the subject is diagnosed with cancer.
 15. Amethod of monitoring the efficacy of a cancer therapy in a humansubject, comprising performing the method of claim 7 at two or moredifferent times on the biological sample, wherein the biological sampleis obtained from, part of, or all of a subject, and whereby the changein strength of the signals characteristic of the gadolinium between thetwo or more different times is correlated with the efficacy of thecancer therapy.
 16. A method of treating a cancer in a subject byneutron capture therapy, comprising: (a) administering to a subjecthaving cancer a composition comprising a compound of claim 1; and (b)radiating the subject with epithermal neutrons; whereby the compoundabsorbs the neutrons and subsequently emits high-energy chargedparticles, thereby treating the cancer.
 17. The method of claim 16,wherein the cancer that is treated is an adult solid tumor or apediatric solid tumor.
 18. The method of claim 17, wherein the cancer isselected from the group consisting of melanoma, neuroblastoma, lungcancer, adrenal cancer, colon cancer, colorectal cancer, ovarian cancer,prostate cancer, liver cancer, subcutaneous cancer, squamous cellcancer, intestinal cancer, retinoblastoma, cervical cancer, glioma,breast cancer, pancreatic cancer, Ewings sarcoma, rhabdomyosarcoma,osteosarcoma, retinoblastoma, Wilms' tumor, and pediatric brain tumors.